NEO-201 is a humanized IgG1 monoclonal antibody (mAb) that reacts to tumor-associated antigens (TAA) derived from pooled allogeneic colon tumor tissue extracts. NEO-201 recognizes tumor-associated variants of CEACAM family members. This mAb is remarkably tumor-specific in its staining profile and reacts to a wide range of human carcinoma cell lines by flow cytometry and tumor tissues by immunohistochemistry. NEO-201 exhibited both ADCC and complement-dependent cellular cytotoxicity (CDC) activity against human carcinoma cell in vitro and counteracted the growth of human pancreatic xenograft tumors in vivo. We investigated an additional mechanism of action of NEO-201 and to further confirm the rationale for clinical testing. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a cell-surface protein expressed by immune cells and tumor cells, and it can inhibit T cell function similar to PD-1 and CTLA-4. CEACAM1 is also a potent inhibitor of natural killer (NK) cell function. Binding between CEACAM1 on NK cells and CEACAM1 or CEACAM5 on tumor cells inhibits activation signaling by NKG2D, which prevents NK cell cytolysis and permits tumor cells to evade NK killing. This study was designed determine whether NEO-201 blocks the CEACAM1 inhibitory pathway to restore antitumor functionality to NK cells. In vitro assays using human tumor cell lines were conducted to identify CEACAM family members bound by NEO-201. Functional assays were conducted to assess the ability of NEO-201 to potentiate the in vitro killing of tumor cells by the NK cell line NK-92, which expresses CEACAM1 and lacks CD16 and the ability to mediate ADCC. NEO-201 was found to react with distinct variants of CEACAM5 and CEACAM6, but not with CEACAM1 or CEACAM8. Expression profiling revealed that various NEO-201+ cell lines expressed differing levels of the native forms of CEACAM5/6 vs. the NEO-201-reactive variant forms of these molecules. Functionally, NEO-201 treatment augmented the cytolytic activity of NK-92 cells against NEO-201+ tumor cells in proportion to their level of CEACAM5 expression (average increase of 2-fold), but not against NEO-201+ cells that only expressed CEACAM6. This study demonstrates that NEO-201 is capable of an additional function. This antibody can block the interaction between tumor cell CEACAM5 and NK cell CEACAM1 to reverse CEACAM1-dependent inhibition of NK cytotoxicity. Experiments are in progress to determine the involvement of NK cell CEACAM1 and/or other checkpoint pathways in this mechanism of action. These results suggest that NEO-201 may potentially reverse CEACAM1-dependent immunosuppression of NK cells in patients whose tumors express the NEO-201-reactive variant of CEACAM5.

Citation Format: Philip Martin Arlen, Massimo Fantini, Justin David, Christina M. Annunziata, Kwong Y. Tsang, Pia Morelli. Mechanisms of action of a neoantigen-targeting antibody NEO-201 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3947.